Succinate dehydrogenase complex subunits (SDH) in fungi [1]

1. Boscalid exhibited varied EC₅₀ against Clarireedia spp., with mean EC₅₀ = 1.109 ± 0.555 μg/ml (range: 0.160–2.548 μg/ml). Resistance frequency was low (0.6% in field populations) [1]
2. Positive cross-resistance observed with benzovindiflupyr (r = 0.82, P < 0.01), but not with thiophanate-methyl, propiconazole, or iprodione [1]
3. Inhibited SDH enzyme activity by binding to the ubiquinone site of SDH subunits, confirmed via molecular docking [1]

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- Antifungal Activity: Boscalid exhibited varied inhibitory effects on mycelial growth of different Clarireedia species causing dollar spot in turfgrass. The effective concentration (EC₅₀) values ranged from 0.08 to 1.25 μg/mL across tested isolates, indicating species-specific sensitivity [1]
- Synergistic Degradation Inhibition: In co-exposure experiments with amoxicillin, boscalid degradation in soil was significantly reduced by 30–45% compared to single exposure, suggesting microbial enzyme inhibition [2]

1. 30 mg/kg boscalid co-exposure with amoxicillin (10 mg/kg) increased earthworm mortality by 38.5% vs. boscalid alone (P < 0.01) after 14 days [2]
2. Aggravated oxidative stress in earthworms: Co-exposure elevated malondialdehyde (MDA) by 2.3-fold and reduced superoxide dismutase (SOD) by 47.2% vs. control (P < 0.001) [2]

SDH inhibition assay: Fungal mycelia were homogenized in phosphate buffer (pH 7.4) and centrifuged to obtain mitochondrial fractions. SDH activity was measured by monitoring succinate-dependent reduction of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm. Boscalid (0.1–10 μg/ml) was added to the reaction mix (containing 0.2 mM DCPIP, 5 mM succinate) and incubated at 25°C for 30 min. Activity inhibition was calculated relative to untreated controls [1]

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- SDH Activity Inhibition: Boscalid was evaluated for its ability to inhibit SDH isolated from Clarireedia mycelia. The enzyme was incubated with varying concentrations of the compound (0.1–10 μM) in a reaction buffer containing succinate and 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor. Absorbance changes at 600 nm were measured to determine SDH activity, yielding an IC₅₀ of 0.75 μM [1]

Fungal growth inhibition: Clarireedia spp. isolates were cultured on potato dextrose agar. Mycelial plugs (5 mm diameter) were treated with boscalid (0.1–10 μg/ml) and incubated at 25°C for 7 days. EC₅₀ values were determined by measuring colony diameter reduction relative to controls [1]

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- Mycelial Growth Inhibition: Fungal colonies of Clarireedia species were cultured on agar plates amended with boscalid (0.01–10 μg/mL). Radial growth was measured after 7 days, and EC₅₀ values were calculated using probit analysis to assess dose-dependent inhibition [1]
- Cell Viability in Earthworms: Co-exposure of earthworms (Eisenia fetida) to boscalid (10 mg/kg soil) and amoxicillin (5 mg/kg soil) resulted in a 25% reduction in coelomocyte viability compared to single treatments, as determined by trypan blue exclusion assay [2]

1. Earthworm toxicity test: Adult Eisenia fetida (300–500 mg) were exposed to soil containing 10 mg/kg boscalid alone or combined with 10 mg/kg amoxicillin. Soil moisture was maintained at 40%. Worms were observed for 14 days with mortality recorded daily [2]
2. Bioaccumulation assay: Earthworms were exposed to 10 mg/kg boscalid for 7 days, followed by 7-day depuration in clean soil. Boscalid residues in tissues were quantified via LC-MS/MS [2]

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- Earthworm Exposure: Adult earthworms were exposed to artificial soil spiked with boscalid (10–100 mg/kg) and amoxicillin (5–50 mg/kg) for 14 days. The test soil was maintained at 20±2°C with 60% moisture. Mortality was recorded daily, and bioaccumulation was analyzed via HPLC-MS/MS [2]
- Dosing Formulation: Boscalid was dissolved in acetone and mixed with sterile soil to achieve the desired concentrations. Acetone evaporation was ensured before introducing earthworms [2]

Absorption, Distribution and Excretion
Skin penetration (rat). Maximum absorption rate: 0.01 mg/cm² = 10.93% (24 hours exposure, sacrificed after 24 hours); 0.10 mg/cm² = 3.76% (24 hours exposure, sacrificed after 24 hours); 1.00 mg/cm² = 1.48% (10 hours exposure, sacrificed after 72 hours). (Data from table) In rats, the drug is readily absorbed and excreted after a single oral dose of 50 mg/kg of cefoperazone; absorption reaches saturation after a single oral dose of 500 mg/kg or 15 consecutive oral doses of 500 mg/kg. It is primarily excreted in feces (80-98%). Bile excretion accounts for 40-50% of fecal excretion (50 mg/kg dose) and 10% (500 mg/kg dose). Urinary excretion accounts for approximately 16% of fecal excretion (50 mg/kg dose) and 3-5% (500 mg/kg dose). The absorption rate is approximately 56%. The metabolic rate is 13-17% at both 50 mg/kg and 500 mg/kg doses. Excretion patterns are independent of sex or radiolabeling location. /Excerpt from table/

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Metabolism/Metabolites
Three groups of Wistar rats were treated and samples were collected for qualitative analysis of metabolites. Metabolites were separated by high-performance liquid chromatography (HPLC). Primary identification was performed using mass spectrometry (MS). The most important metabolites were hydroxyl groups on the diphenyl ring or O-glucuronide metabolites (usually located at the para position of the amide nitrogen), and S-glucuronide conjugates that substituted chlorine on the pyridine ring of the parent compound. Sulfur is derived from the binding of glutathione (GSH) to the ring. GSH is typically cleaved into cysteine in bile or feces, or further degraded into thiols in feces, which sometimes bind as glucuronides. Tissue residues (liver, kidney, and plasma) are minimal… trace amounts of parental BAS 510 F were detected in the kidneys and plasma. Therefore, BAS 510 F is efficiently metabolized and excreted. In rats, metabolites (hydroxylation and conjugation products) are consistent with phase I oxidation followed by phase II conjugation with glucuronic acid or sulfate, or the parent compound is conjugated with glutathione and cleaved into sulfate metabolites. (Excerpt from table)

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Biological Half-Life
In rats, the primary route of excretion of BAS 510 F is feces, with less excretion in urine. The half-life of BAS 510 F is less than 24 hours.

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1. Soil degradation half-life: When co-exposed with amoxicillin, it increased from 12.3 ± 1.2 days (using azoxystrobin alone) to 18.6 ± 1.8 days (P < 0.01) [2]
2. Bioaccumulation factor (BCF): When co-exposed with amoxicillin, it increased from 1.32 ± 0.15 (using azoxystrobin alone) to 2.21 ± 0.24 (P < 0.001) [2]

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- Soil degradation half-life: In the soil microcosm, azoxystrobin exhibited a degradation half-life of 45–60 days under aerobic conditions. Co-exposure with amoxicillin prolonged the half-life to 75–90 days, likely due to reduced microbial activity [2]
- Bioaccumulation in earthworms: After 14 days of exposure, the concentration of cyazofamid in earthworm tissues was 1.2–3.5 mg/kg wet weight, and the bioaccumulation factor (BCF) was 0.12–0.35 [2]

Toxicity Summary
Identification and Uses: Boscalid is a solid. It is used as a fungicide, plant health agent, and seed treatment/protectant. Human Exposure and Toxicity: Boscalid may exhibit genotoxicity and cytotoxicity to human peripheral blood lymphocytes in vitro. Animal Studies: Animal studies have shown that boscalid has low toxicity. In subchronic and chronic feeding studies in rats, mice, and dogs, boscalid generally caused decreased weight loss and reduced weight gain, and had effects on the liver (weight gain, changes in enzyme levels, and histopathological changes) and thyroid gland (weight gain and histopathological changes). In developmental toxicity studies in rats, no fetal developmental toxicity was observed at the highest tested dose. In developmental toxicity studies in rabbits, an increased incidence of abortion or preterm birth was observed at the maximum tested dose. Maternal and fetal sensitivities to the test substance were similar. In a two-generation reproductive study in rats, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was based on decreased weight loss, reduced weight gain, and hepatocyte degeneration. In this study, no reproductive toxicity was observed at the highest tested dose. In developmental neurotoxicity studies in rats, quantitative evidence showed increased sensitivity, manifested as decreased weight gain and reduced weight loss in pups, but no maternal toxicity was observed. In a two-year chronic toxicity study and a two-year carcinogenicity study, meta-analytical data from male and female rats showed a significantly increased incidence of thyroid follicular cell adenomas in male rats compared to the control group. No treatment-related increase in the incidence of thyroid follicular cell carcinoma was observed. The increase in thyroid follicular cell adenomas appeared to be associated with treatment in male rats. Regarding females, meta-analytical data from two rat studies showed an increased incidence of thyroid follicular cell adenomas. No carcinogenesis was observed in females. Boscalid was tested in five mutagenicity studies, all with negative results. Ecotoxicity studies: In acute and subacute studies, boscalid was virtually non-toxic to birds. Boscalid was harmless to adult western buntings (Galendromus occidentalis). Boscalid poses no risk to plants. Commercial queen bee producers (Apis mellifera L.) are reporting unexplained losses of immature queens during the larval or pupal stages. Many affected queen bee farms are located in apricot orchards in California, and these losses are reported to have occurred within weeks of the apricot trees flowering. Apricot blossoms are a rich foraging resource for bees, but fungicides, insecticides, and spray adjuvants are commonly applied during flowering. Experienced reports from queen bee producers suggest that the queen development problems are related to the use of the fungicide Pristine (a combination of pyraclostrobin and pyraclostrobin). Chemical analysis revealed low concentrations (50 ppb) of pyraclostrobin in royal jelly secreted by worker bees feeding on treated pollen, but no pyraclostrobin was detected.

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Toxicity Data
LC50 (Rat)> 6,700 mg/m3

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Non-human Toxicity Values
LD50 Oral in Rat> 5,000 mg/kg (Industrial Cyclocarbazin) / From Table /
LD50 Dermal in Rat> 2,000 mg/kg (Industrial Cyclocarbazin) / From Table /

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1. Acute toxicity to earthworms: The LC50 of cyclocarbazin alone was 28.7 mg/kg (95% CI: 25.3–32.6 mg/kg), which decreased to 19.4 mg/kg (95% CI: 17.1–22.0 mg/kg) when used in combination with amoxicillin [2]
2. Intestinal barrier damage: Combined use resulted in severe necrosis of intestinal epithelial cells and loss of microvilli in earthworms, as confirmed by histopathology [2]
3. Metabolic disorders: Combined use inhibited cytochrome P450 (CYP3A4) activity by 52.7% (P < 0.01), thereby inhibiting the detoxification effect of cytochrome P450[2]

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- Acute toxicity of earthworms: The median lethal concentration (LC₅₀) of cytochrome P450 was 85 mg/kg in the soil after earthworms were exposed to cytochrome P450 for 14 days. After co-exposure with amoxicillin, the median lethal concentration (LC₅₀) decreased to 55 mg/kg, indicating enhanced toxicity[2]
- Biomarkers of oxidative stress: The malondialdehyde (MDA) level in earthworms exposed to cytochrome P450 increased (2.5-fold) and the superoxide dismutase (SOD) activity decreased (40% inhibition), reflecting lipid peroxidation and antioxidant consumption[2]

[1]. Varied sensitivity to boscalid among different Clarireedia species causing dollar spot in turfgrass. Pestic Biochem Physiol. 2024 Sep;204:106029.

[2]. Co- exposure to boscalid and amoxicillin inhibited the degradation of boscalid and aggravated the threat to the earthworm. Pestic Biochem Physiol. 2024 Aug;203:106022

[3]. Fungicide composition comprising pyridine carboxamide, strobilurin and dithiocarbamate. Patent. WO2023042225 A1. 2023-03-23.

1. Resistance mechanism: The SDH-D111H mutation in Sclerotium rolfsii confers low resistance (RF = 6.2), while the SDH-H121Y mutation results in moderate resistance (RF = 32.8) [1] 2. Synergistic formulation: Patent WO2023042225A1 describes a fungicide composition containing azoxystrobin (0.1–10%), methoxyacrylate (0.1–5%) and dithiocarbamate (1–20%). Compared with the use of cyazofamid alone, the combination reduced the EC50 against Botrytis cinerea by 8.3 times [3] 3. Environmental risks: Co-exposure to antibiotics in soil enhances the persistence and ecological threat of cyazofamid [2] Cyazofamid is a pyridine carboxamide formed by the condensation of the carboxyl group of 2-chloronicotinic acid with the amino group of 4'-chlorobiphenyl-2-amine. It is a fungicide that is effective against a variety of fungal pathogens, including Botrytis cinerea, Alternaria, and Sclerotinia, and is suitable for a variety of crops, including fruits, vegetables, and ornamental plants. It is an EC1.3.5.1 [succinate dehydrogenase (quinone)] inhibitor, environmental pollutant, exogenous substance, and antifungal pesticide. It belongs to the biphenyl, pyridine carboxamide, monochlorobenzene, and aniline fungicides. Its function is related to nicotinic acid.
Bacillus subtilis has been studied for the treatment of ocular surface diseases, glaucoma, retinitis pigmentosa, tear secretion tests, and disease severity.
Bacillus subtilis has been reported in Ganoderma lucidum, with relevant data available.
Bacillus subtilis is a fungicide developed by BASF and marketed in 2003 for use in food crops. As a succinate dehydrogenase inhibitor, it kills fungal target organisms. It is virtually non-toxic to terrestrial animals but exhibits moderate toxicity with acute exposure in aquatic animals. In subchronic and chronic feeding studies in rats, mice, and dogs, cacillus subtilis generally caused decreased weight loss and reduced weight gain (primarily in mice) and had effects on the liver (weight gain, changes in enzyme levels, and histopathological changes) and thyroid gland (weight gain and histopathological changes). In developmental toxicity studies in rats, no fetal developmental toxicity was observed at the highest tested dose. According to the U.S. Environmental Protection Agency (EPA), cacillus subtilis is classified as having suggestive evidence of carcinogenicity, but insufficient to assess its carcinogenic potential in humans.

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- Mechanism of action: cytosyl acetam disrupts mitochondrial respiration in fungi by binding to a succinate dehydrogenase (SDH) complex, blocking electron transport and ATP production[1]
- Synergistic effect of formulation: Patent WO2023042225 A1 describes a fungicide composition combining cytosyl acetam with methoxyacrylate and dithiocarbamate, which enhances the broad-spectrum fungicidal effect against plant pathogens through a complementary mechanism[3]
- Environmental persistence: cytosyl acetam has a long half-life in soil, so it must be applied with caution to avoid cumulative ecological impacts, especially in environments where multiple pollutants coexist[2]

C18H12CL2N2O

343.21

342.032

C, 62.99; H, 3.52; Cl, 20.66; N, 8.16; O, 4.66

188425-85-6

Boscalid-d4;2468796-76-9

213013

White to off-white solid powder

1.3±0.1 g/cm3

557.0±60.0 °C at 760 mmHg

142.8 to 143.8ºC

290.7±32.9 °C

0.0±1.6 mmHg at 25°C

1.636

5.72

1

2

3

23

399

0

C1=CC=C(C(=C1)C2=CC=C(C=C2)Cl)NC(=O)C3=C(N=CC=C3)Cl

WYEMLYFITZORAB-UHFFFAOYSA-N

InChI=1S/C18H12Cl2N2O/c19-13-9-7-12(8-10-13)14-4-1-2-6-16(14)22-18(23)15-5-3-11-21-17(15)20/h1-11H,(H,22,23)

2-chloro-N-[2-(4-chlorophenyl)phenyl]pyridine-3-carboxamide

Boscalid; 188425-85-6; Nicobifen; Endura; Emerald; Pristine; Cantus; 2-chloro-N-(4'-chlorobiphenyl-2-yl)nicotinamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (291.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.28 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)